Structural and functional characterisation of the methionine adenosyltransferase from Thermococcus kodakarensis
- Julia Schlesier†1,
- Jutta Siegrist†2,
- Stefan Gerhardt1,
- Annette Erb2,
- Simone Blaesi2,
- Michael Richter3,
- Oliver Einsle1, 4 and
- Jennifer N Andexer2Email author
© Schlesier et al.; licensee BioMed Central Ltd. 2013
Received: 2 July 2013
Accepted: 11 October 2013
Published: 18 October 2013
Methionine adenosyltransferases catalyse the synthesis of S-adenosylmethionine, a cofactor abundant in all domains of life. In contrast to the enzymes from bacteria and eukarya that show high sequence similarity, methionine adenosyltransferases from archaea diverge on the amino acid sequence level and only few conserved residues are retained.
We describe the initial characterisation and the crystal structure of the methionine adenosyltransferase from the hyperthermophilic archaeon Thermococcus kodakarensis. As described for other archaeal methionine adenosyltransferases the enzyme is a dimer in solution and shows high temperature stability. The overall structure is very similar to that of the bacterial and eukaryotic enzymes described, with some additional features that might add to the stability of the enzyme. Compared to bacterial and eukaryotic structures, the active site architecture is largely conserved, with some variation in the substrate/product-binding residues. A flexible loop that was not fully ordered in previous structures without ligands in the active side is clearly visible and forms a helix that leaves an entrance to the active site open.
The similar three-dimensional structures of archaeal and bacterial or eukaryotic methionine adenosyltransferases support that these enzymes share an early common ancestor from which they evolved independently, explaining the low similarity in their amino acid sequences. Furthermore, methionine adenosyltransferase from T. kodakarensis is the first structure without any ligands bound in the active site where the flexible loop covering the entrance to the active site is fully ordered, supporting a mechanism postulated earlier for the methionine adenosyltransferase from E. coli. The structure will serve as a starting point for further mechanistic studies and permit the generation of enzyme variants with different characteristics by rational design.
KeywordsS-Adenosylmethionine synthase S-Adenosylmethionine Thermostable enzyme Archaea
In the following we present the structural and functional characterisation of MAT from Thermococcus kodakarensis (also T. kodakaraensis), a new member of the group of archaeal MATs. The sulphur-reducing, hyperthermophilic archaeon T. kodakarensis is classified in the phylum euryarchaeota and is found in many high-temperature environments. It grows optimally at a temperature of 85°C and is able to survive up to 100°C . Since 2005, when the genome sequence of T. kodakarensis was determined , many enzymes involved in several pathways were investigated .
Optimisation of the HPLC assay
Cloning and initial characterisation of Tk MAT
Kinetic parameters of MATs from different domains of life
V max [μmol/min/mg]
1.95 ± 0.26
K M (Met) [mM]
0.31 ± 0.07
K M (ATP) [mM]
6.54 ± 2.49
HPLC (SCX column)
radioactive filter binding assay
radioactive filter binding assay
radioactive filter binding assay
0.1 M Tris–HCl, 0.02 M MgCl2, 0.2 KCl, pH 8.0
50 mM K-Hepes, 10 mM MgCl2, 25 mM KCl, pH 8.0
0.1 Tris-Cl, 6 mM MgCl2, 0.1 KCl, pH 8.3
50 mM K-Tes, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, 0.3 mM Na2EDTA, 0.1 mM BSA, pH 7.4
10 mM l-methionine 10 mM ATP
0.1 mM l-[methyl-14C]-methionine 5 mM ATP
0.6 mM l-[methyl-14C]-methionine 4.4 mM 14C-ATP
0.01 mM l-[methyl-14C]-methionine 5 mM ATP
Graham et al. 
Markham et al. 
Kotb & Kredich 
Overall structure of Tk MAT
r.m.s.d. values for MATs from different domains of life
Similarities and differences in the active site
The amino acids involved in the stabilisation of the triphosphate are divers between different MATs. That, and the fact that the ligands bound derive from different steps of the reaction (ATP, triphosphate, (diphosphono)aminophosphonic acid, pyrophosphate, phosphate), makes a comparison imprecise.
Access to the active site
The high thermostability observed for Tk MAT matches the optimal growth temperature of the hyperthermophilic archaeon T. kodakarensis very well; similar behaviour was described for the homologous enzymes from S. solfataricus and M. jannaschii [14, 15]. The kinetic parameters obtained are in a similar range to those reported for other MATs, only the KM value for ATP is significantly higher. However, the assays as well as the reaction conditions used in the single experiments, differ widely making a direct comparison difficult (Table 1).
The additional β-strand in the C-terminal domain is also present in the corresponding S. solfataricus structure, but was not represented in the homology model for Mj MAT, as this was based on the human structure where the β-strand is absent . However, the first 15 and 16 amino acids of the enzymes from human and rat, respectively, were not ordered in the corresponding crystal structures, so that the presence of the additional β-strand in solution cannot be excluded in these enzymes. In archaeal MATs the additional β-strand extends the β-sheet of the C-terminal domain to match the four-stranded sheets found in the N-terminal and central domains, and therefore might add to the higher stability of archaeal MATs by providing a direct connection between the N- and C-termini of the peptide chain within a stable secondary structure. The extended β-sheet in the central domain might also exhibit stabilisation effects by wrapping around the loops at the edge of the N-terminal domain (see Additional file 1).
A major difference between Tk MAT and other MAT structures without any bound ligands is that the helix covering the active site is completely defined in the structure. One exception to this is a structure of Ec MAT that was crystallised at low temperatures (PDB-ID 1FUG) . Here, the loop was modelled in a quasi-closed conformation but does not show the ordered helical secondary structure visible in Ec MAT structures with bound SAM (PDB-ID 1RG9, 1P7L). Until now the flexible loop was thought to be disordered in the open conformation, however all four monomers in the asymmetric unit of the Tk MAT crystal showed the same ordered helix in the open structure. The higher intrinsic stability of the thermophilic enzyme could explain why this region is well defined in the structure of Tk MAT. However, in the corresponding open structure from S. solfataricus (PDB-ID 4HPV), another thermophilic organism, the loop is not defined.
Our data supports the mechanism of substrate binding described for Ec MAT where residues from the flexible loop form their respective interactions with the substrates after they have entered the active site along with movement of the flexible helix into the closed position . The position of these residues (Leu145 [Tk MAT], Ile 102 [Ec MAT] stabilising the methionine side chain, and Asp144 [Tk MAT], Arg229 [Ec MAT] interacting with the adenine amino group) are clearly different in the open and closed structures (Figure 5). Some of the active site residues responsible for substrate/product binding in Ec MAT and other bacterial and eukaryotic MATs, including the putatively catalytic histidine, are conserved in Tk MAT, while some functional groups appear to be stabilised in a different manner. A closer inspection of the active site based on co-crystallised substrates, products or inhibitors will show if these differences are responsible for the variation in the substrate range described for the archaeal MAT from M. jannaschii, in comparison to Ec MAT .
We present the structure of a thermophilic, archaeal MAT that displays several novel features in comparison to MATs from bacteria or eukarya, including extended β-sheets that may be responsible for the increased stability of MATs from thermophilic organisms. SAM is an important cofactor for a wide range of enzymatic reactions. Enzymes from thermophilic organisms are often used as biocatalysts for technical applications due to their high stability . Commercially available SAM is usually extracted from yeast, however for some applications such as the generation of SAM-derivatives or isotope-labelled compounds using isolated enzymes might be advantageous. The structure described here will serve as a basis for the rational design of MAT variants to further extend the substrate range.
Cloning of Tk MAT
The 1155 bp gene of the MAT was amplified by PCR from genomic DNA of T. kodakarensis using the following primers: 5′-TATATATACATATG GCAAAACACCTTTTTACGTCCG-3′ and 5′-TATACTCGAG TTACTTCAGACCGGCAGCAT-3′. After restriction with the appropriate restriction enzymes (Nco I and Xho I, respectively), the fragments were ligated into the pET28a(+) vector finally coding for the MAT carrying an N-terminal His-tag, and E. coli BL21(DE3)-CodonPlus-RP competent cells were transformed with the construct.
Expression and purification of Tk MAT
Cells were grown in 500 mL LB Lennox medium supplemented with 34 mg/L chloramphenicol and 50 mg/L kanamycin. Expression was induced by addition of isopropyl thiogalactoside (IPTG, final concentration 0.2 mM) at an optical density (OD600) of 0.6. After incubation (4 h, 180 rpm, 37°C) the cells were harvested, resuspended in purification buffer (40 mM Tris-HCl, 100 mM NaCl, pH 8.0) and disrupted by one passage through an EmulsiFlex-B30 homogenizer (Avestin). The enzyme was purified via Ni-NTA affinity chromatography. After washing with purification buffer containing 0 and 100 mM imidazole, Tk MAT was eluted from the column with with 500 mM imidazole in purification buffer. The protein was desalted on PD-10 columns.
The heterologous expression of the SeMet derivative of Tk MAT was performed as described by Dias et al. . A preculture of E. coli BL21(DE3)RP/pET28a-TkMAT in minimal medium (6 g Na2HPO4, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl, 1 mM MgSO4, 4 g glucose and 0.5 mg thiamine per 980 mL, supplemented with with each 50 mg/L kanamycin and chloramphenicol) was grown for 14 h at 37°C and 180 rpm. The main culture (minimal medium, 500 mL) was incubated at 37°C and 250 rpm. At an OD600 of 0.325 an amino acid mix (0.1 g l-lysine, 0.1 g l-phenylalanine, 0.1 g l-threonine, 50 mg l-leucine, 50 mg l-isoleucine, 50 mg l-valine and 50 mg l-selenomethionine in 10 mL 0.5 M HCl, sterile filtrated) and a corresponding amount of NaOH were added and incubated for 30 min, then the expression was induced with IPTG (end concentration 0.2 mM) for 24 h at 16°C and 200 rpm. The protein was purified in the same way as described for Tk MAT.
Size exclusion chromatography
2 mL of the protein solution (5 mg/mL) were loaded on a gel filtration column (Superdex 200 prep grade beads in an XK 16/70 glass column [GE Healthcare]) equilibrated with 40 mM Tris-HCl, 150 mM NaCl, pH 8.0 and eluted at a flow rate of 1 mL/min. To estimate the native size of the protein the column was calibrated with a mixture of proteins of known size.
The activity of the MAT was determined in 0.1 M Tris-HCl, 0.02 M MgCl2, 0.2 M KCl, pH 8.0 using varying amounts of l-methionine and ATP (each 10 mM for standard assays). Assays were performed with 0.5 mg Tk MAT in 1 mL end volume at 37°C. The enzyme reaction was stopped after 3 min by the addition of 2% (v/v) HClO4 and subsequently neutralised with NaOH. Separation of products was achieved by HPLC (Agilent 1100 Series) using a Sphere-Image 5 SCX column, (250 × 4.6 mm). The mobile phase used was as described by Kamarthapu et al.  using a flow rate of 1.5 mL/min. Standards were dissolved in the enzyme reaction buffer described above. Kinetic parameters were calculated by non-linear fitting using the Origin software.
Circular dichroism (CD) spectroscopy
CD spectra were measured with a Jasco J-810 Spectropolarimeter. 1 μM enzyme in 3 mL HPLC assay buffer were heated from 20°C to 100°C in 0.1°C steps and the CD spectrum was measured at 224 nm.
Tk MAT was crystallised using the sitting drop vapour diffusion method. 1 μL of protein solution (10 mg/mL) was mixed with the same volume of a reservoir solution and equilibrated against 500 μL of the same reservoir in sealed Cryschem 24-1 SBS plates. SeMet-labelled protein crystallised as octahedral crystals belonging to the tetragonal space group P43, using a reservoir solution containing 22% (w/v) polyethylene glycol 3350 and 0.2 M sodium citrate at room temperature. A different, rhombic crystal morphology was obtained from a condition containing 50 mM HEPES/NaOH buffer at pH 7.5, 35% pentaerythritol propoxylate, 3% (w/v) of sucrose and 0.2 M KCl.
In order to facilitate cryo-protection, the crystals were dehydrated by addition of an additional 500 μL of PEG 3350 (50%) to the reservoir on the day before harvesting. Crystals were mounted into nylon loops and flash-cooled in liquid nitrogen. Diffraction data sets were collected at the Swiss Light Source (Paul-Scherrer-Institut, Villigen, Switzerland) either at beam line X06DA with a Pilatus 2M detector or at beam line X06SA with a Pilatus 6M detector (Dectris). 360° of data were collected in steps of 0.5° per image. For phase determination by single-wavelength anomalous dispersion (SAD), a data set was collected at the peak wavelength of the selenium K-edge at 0.9796 Å. The octahedrally-shaped crystals of the SeMet derivative diffracted to 3.8 Å resolution. The C 2 crystal form was used to collect a native data set to a limiting resolution of 2.0 Å.
Data processing and structure solution
Data collection and refinement statistics
PDB accession code
Unit cell parameters
a, b, c [Å]
121.2, 121.2, 247.1
134.8, 57.8, 236.4
α, β, γ [°]
90.0, 90.0, 90.0
90.0, 104.0, 90.0
Monomers per a.u.
50.0 – 3.8 (3.9 – 3.8)*
50.0 – 2.0 (2.1 – 2.0)*
No. of unique reflections
est. coordinate error [Å]
r.m.s.d. bond lengths [Å]
r.m.s.d. bond angles [°]
average B-factor [Å 2 ]
Figures were generated using PyMOL (Schrödinger LLC), interactions of ligands with active site residues are based on the corresponding structures using the PoseView application .
MAT from Escherichia coli
MAT from Homo sapiens
MAT from Methanocaldococcus jannaschii
Single wavelength anomalous dispersion
Strong cation exchange
MAT from Sulfolobus solfataricus
MAT from Thermococcus kodakarensis.
The authors would like to thank Dr. Rafael Say and Prof. Georg Fuchs (University of Freiburg) for the genomic DNA of T. kodakarensis, Simon Aschwanden (Laboratory for Biomaterials, EMPA) for help with activity measurements, Dr. Daniel Wohlwend for help with gel filtration analysis, and Prof. Michael Müller (University of Freiburg) for useful input. Data was collected on beamline X06DA at the Swiss Light Source, Paul-Scherrer-Institut, Villigen, CH. We thank the beamline staff for their usual excellent assistance during data collection. This work was supported by the European Research Council (to OE). The article processing charge was funded by the German Research Foundation (DFG) and the Albert-Ludwigs-University Freiburg in the funding programme Open Access Publishing.
- Fontecave M, Atta M, Mulliez E: S-adenosylmethionine: nothing goes to waste. Trends Biochem Sci 2004, 29: 243–249. 10.1016/j.tibs.2004.03.007View ArticlePubMedGoogle Scholar
- Frey PA, Hegeman AD, Ruzicka FJ: The Radical SAM Superfamily. Crit Rev Biochem Mol Biol 2011, 43: 63–88.View ArticleGoogle Scholar
- Kim J, Xiao H, Bonanno JB, Kalyanaraman C, Brown S, Tang X, Al-Obaidi NF, Patskovsky Y, Babbitt PC, Jacobson MP, Lee Y-S, Almo SC: Structure-guided discovery of the metabolite carboxy-SAM that modulates tRNA function. Nature 2013, 498: 123–126. 10.1038/nature12180PubMed CentralView ArticlePubMedGoogle Scholar
- Sánchez-Pérez GF, Bautista JM, Pajares MA: Methionine Adenosyltransferase as a Useful Molecular Systematics Tool Revealed by Phylogenetic and Structural Analyses. J Mol Biol 2004, 335: 693–706. 10.1016/j.jmb.2003.11.022View ArticlePubMedGoogle Scholar
- Reczkowski RS, Taylor JC, Markham GD: The Active-Site Arginine of S-Adenosylmethionine Synthetase Orients the Reaction Intermediate. Biochemistry 1998, 37: 13499–13506. 10.1021/bi9811011View ArticlePubMedGoogle Scholar
- Kamarthapu V, Rao KV, Srinivas PNBS, Reddy GB, Reddy VD: Structural and kinetic properties of Bacillus subtilis S-adenosylmethionine synthetase expressed in Escherichia coli. Biochim Biophys Acta 2008, 1784: 1949–1958. 10.1016/j.bbapap.2008.06.006View ArticlePubMedGoogle Scholar
- Zhao X, Gust B, Heide L: S-Adenosylmethionine (SAM) and antibiotic biosynthesis: effect of external addition of SAM and of overexpression of SAM biosynthesis genes on novobiocin production in Streptomyces. Arch Microbiol 2010, 192: 289–297. 10.1007/s00203-010-0548-xView ArticlePubMedGoogle Scholar
- Luo Y, Yuan Z, Luo G, Zhao F: Expression of Secreted His-Tagged S-adenosylmethionine Synthetase in the Methylotrophic Yeast Pichia pastoris and Its Characterization, One-Step Purification, and Immobilization. Biotechnol Prog 2008, 24: 214–220. 10.1021/bp0702727View ArticlePubMedGoogle Scholar
- González B, Pajares MA, Hermoso JA, Guillerm D, Guillerm G, Sanz-Aparicio J: Crystal Structures of Methionine Adenosyltransferase Complexed with Substrates and Products Reveal the Methionine-ATP Recognition and Give Insights into the Catalytic Mechanism. J Mol Biol 2003, 331: 407–416. 10.1016/S0022-2836(03)00728-9View ArticlePubMedGoogle Scholar
- Ramani K, Yang H, Kuhlenkamp J, Tomasi L, Tsukamoto H, Mato JM, Lu SC: Changes in the expression of methionine adenosyltransferase genes and S-adenosylmethionine homeostasis during hepatic stellate cell activation. Hepatology 2010, 51: 986–995.PubMed CentralPubMedGoogle Scholar
- Shafqat N, Muniz JRC, Pilka ES, Papagrigoriou E, von Delft F, Oppermann U, Yue WW: Insight into S-adenosylmethionine biosynthesis from the crystal structures of the human methionine adenosyltransferase catalytic and regulatory subunits. Biochem J 2013, 452: 27–36.PubMed CentralView ArticlePubMedGoogle Scholar
- Markham GD, Hafner EW, Tabor CW, Tabor H: S-Adenosylmethionine synthetase from Escherichia coli. J Biol Chem 1980, 255: 9082–9092.PubMedGoogle Scholar
- Komoto J, Yamada T, Takata Y, Markham GD, Takusagawa F: Crystal Structure of the S-Adenosylmethionine Synthetase Ternary Complex: A Novel Catalytic Mechanism of S-Adenosylmethionine Synthesis from ATP and Met. Biochemistry 2004, 43: 1821–1831. 10.1021/bi035611tView ArticlePubMedGoogle Scholar
- Porcelli M, Cacciapuoti G, Carteni-Farina M, Gambacorta A: S-Adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus. Eur J Biochem 1988, 177: 273–280. 10.1111/j.1432-1033.1988.tb14373.xView ArticlePubMedGoogle Scholar
- Lu ZJ, Markham GD: Enzymatic Properties of S-Adenosylmethionine Synthetase from the Archaeon Methanococcus jannaschii. J Biol Chem 2002, 277: 16624–16631. 10.1074/jbc.M110456200View ArticlePubMedGoogle Scholar
- Graham DE, Bock CL, Schalk-Hihi C, Lu ZJ, Markham GD: Identification of a Highly Diverged Class of S-Adenosylmethionine Synthetases in the Archaea. J Biol Chem 2000, 275: 4055–4059. 10.1074/jbc.275.6.4055View ArticlePubMedGoogle Scholar
- Littlechild JA: Thermophilic archaeal enzymes and applications in biocatalysis. Biochem Soc Trans 2011, 39: 155–158. 10.1042/BST0390155View ArticlePubMedGoogle Scholar
- Egorova K, Antranikian G: Industrial relevance of thermophilic Archaea. Curr Opin Microbiol 2005, 8: 649–655. 10.1016/j.mib.2005.10.015View ArticlePubMedGoogle Scholar
- Garrido F, Alfonso C, Taylor JC, Markham GD, Pajares MA: Subunit association as the stabilizing determinant for archaeal methionine adenosyltransferases. Biochim Biophys Acta 2009, 1794: 1082–1090. 10.1016/j.bbapap.2009.03.018PubMed CentralView ArticlePubMedGoogle Scholar
- Garrido F, Taylor JC, Alfonso C, Markham GD, Pajares MA: Structural basis for the stability of a thermophilic methionine adenosyltransferase against guanidinium chloride. Amino Acids 2012, 42: 361–373. 10.1007/s00726-010-0813-yPubMed CentralView ArticlePubMedGoogle Scholar
- Atomi H, Fukui T, Kanai T, Morikawa M, Imanaka T: Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1. Archaea 2004, 1: 236–267.View ArticleGoogle Scholar
- Fukui T, Atomi H, Kanai T, Matsumi R, Fujiwara S, Imanaka T: Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res 2005, 15: 352–363. 10.1101/gr.3003105PubMed CentralView ArticlePubMedGoogle Scholar
- Atomi H, Imanaka T, Fukui T: Overview of the genetic tools in the Archaea. Front Microbiol 2012, 3: 1–13.View ArticleGoogle Scholar
- Markham GD, Pajares MA: Structure-function relationships in methionine adenosyltransferases. Cell Mol Life Sci 2008, 66: 636–648.View ArticleGoogle Scholar
- Kotb M, Kredich NM: S-Adenosylmethionine synthetase from human lymphocytes. Purification and characterization. J Biol Chem 1985, 260: 3923–3930.PubMedGoogle Scholar
- Takusagawa F, Kamitori S, Markham GD: Structure and Function of S-Adenosylmethionine Synthetase: Crystal Structures of S-Adenosylmethionine Synthetase with ADP, BrADP, and PPi at 2.8 Å Resolution. Biochemistry 1996, 35: 2586–2596. 10.1021/bi952604zView ArticlePubMedGoogle Scholar
- Fu Z, Hu Y, Markham GD, Takusagawa F: Flexible Loop in the Structure of S-Adenosylmethionine Synthetase Crystallized in the Tetragonal Modification. J Biomol Struct Dyn 1996, 13: 727–739. 10.1080/07391102.1996.10508887View ArticlePubMedGoogle Scholar
- Dias MVB, Huang F, Chirgadze DY, Tosin M, Spiteller D, Dry EFV, Leadlay PF, Spencer JB, Blundell TL: Structural Basis for the Activity and Substrate Specificity of Fluoroacetyl-CoA Thioesterase FlK. J Biol Chem 2010, 285: 22495–22504. 10.1074/jbc.M110.107177PubMed CentralView ArticlePubMedGoogle Scholar
- Kabsch W: XDS. Acta Crystallogr D 2010, 66: 125–132. 10.1107/S0907444909047337PubMed CentralView ArticlePubMedGoogle Scholar
- Evans P: An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D 2011, 67: 282–292. 10.1107/S090744491003982XPubMed CentralView ArticlePubMedGoogle Scholar
- Cowtan K, Emsley P, Wilson KS: From crystal to structure with CCP4. Acta Crystallogr D 2011, 67: 233–234. 10.1107/S0907444911007578PubMed CentralView ArticlePubMedGoogle Scholar
- Emsley P, Lohkamp B, Scott WG, Cowtan K: Features and development of Coot. Acta Crystallogr D 2010, 66: 486–501. 10.1107/S0907444910007493PubMed CentralView ArticlePubMedGoogle Scholar
- Murshudov GN, Vagin AA, Dodson EJ: Refinement of Macromolecular Structures by the Maximum-Likelihood Method. Acta Crystallogr D 1997, 53: 240–255. 10.1107/S0907444996012255View ArticlePubMedGoogle Scholar
- Stierand K, Rarey M: Drawing the PDB: Protein - Ligand Complexes in Two Dimensions. ACS Med Chem Lett 2010, 1: 540–545. 10.1021/ml100164pPubMed CentralView ArticlePubMedGoogle Scholar
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